GSM7119241: SY5609_45_R1 [PRO-seq]; Homo sapiens; OTHER
RNA-seq: Cells were then harvested by washing with cold D-PBS, scraped in D-PBS into 15mL conical tubes, and centrifuged at 1000rpm for 5min 4C. RNA was extracted with TRIzol according to the manufacturer's instructions. DNaseI digestion was then performed with the RNeasy kit (Qiagen) using the on-column protocol (Rimel et al., 2020). RNA was eluted in 50µL RNase-free water and samples analyzed by Qubit and TapeStation. PRO-seq: As previously described in (Levandowski, C. B.; Jones, T.; Gruca, M.; Ramamoorthy, S.; Dowell, R. D.; Taatjes, D. J, 2021), after treatment the cells were washed 3x with ice cold PBS, and then treated with 10 ml (per 15 cm plate) ice-cold lysis buffer (10 mM Tris–HCl pH 7.4, 2 mM MgCl2, 3 mM CaCl2, 0.5% NP-40, 10% glycerol, 1 mM DTT, 1x Protease Inhibitors (1mM Benzamidine (Sigma B6506-100G), 1mM Sodium Metabisulfite (Sigma 255556-100G), 0.25mM Phenylmethylsulfonyl Fluoride (American Bioanalytical AB01620), and 4U/mL SUPERase-In). Cells were centrifuged with a fixed-angle rotor at 1000xg for 15 min at 4◦C. Supernatant was removed and pellet was resuspended in 1.5 mL lysis buffer to a homogenous mixture by pipetting 20-30X before adding another 8.5 mL lysis buffer. Suspension was centrifuged with a fixed-angle rotor at 1000xg for 15 min at 4◦C. Supernatant was removed and pellet was resuspended in 1 mL of lysis buffer and transferred to a 1.7 mL pre-lubricated tube (Costar cat. No. 3207). Suspensions were then pelleted in a microcentrifuge at 1000xg for 5 min at 4◦C. Next, supernatant was removed and pellets were resuspended in 500 μL of freezing buffer (50 mM Tris pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 4U/ml SUPERase-In). Nuclei were centrifuged 2000xg for 2 min at 4◦C. Pellets were resuspended in 100 μL freezing buffer. To determine concentration, nuclei were counted from 1 μL of suspension and freezing buffer was added to generate 100 μL aliquots of 10x106 nuclei. Aliquots were flash frozen in liquid nitrogen and stored at −80◦C. RNA-seq: Samples were concentrated, 300-400ng/µL, and of high quality, RIN values >8.6. For deep mRNA sequencing to asses splicing alterations, poly(A) selection was performed with the KAPA mRNA HyperPrep Kit with 1µg of RNA containing 1:100 diluted ERCC RNA spike-in mix (Invitrogen Thermo Fisher). KAPA mRNA HyperPrep Kit, NovaSEQ6000, using a paired-end 150- bp cycle (2x150). PRO-seq: Nuclear run-on experiments were performed as described previously (Mahat, D. B., Kwak, H., Booth, G. T., Jonkers, I. H., Danko, C. G., Patel, R. K., et al. (2016). Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nature Protocols, 11(8), 1455–1476. with the following modifications: the final concentration of non-biotinylated CTP was raised from 0.25 μM to 25 μM, a clean-up and size selection was performed using 1X AMPure XP beads (1:1 ratio) (Beckman) prior to test PCR and final PCR amplification, and the final library clean-up and size selection was accomplished using 1X AMPure XP beads (1:1 ratio) (Beckman).